Isopentenyl diphosphate (IPP) and the isomeric compound, dimethylallyl diphosphate (DMAPP) are the fundamental building blocks of isoprenoids in all organisms. The isoprenoids include more than 23,000 naturally occurring molecules of both primary and secondary metabolism (Sacchettiii, J. C. & Poulter, C. D., 1997). The chemical diversity of this natural product class reflects their wide-ranging physiological roles in all living systems (Connolly, J. D. & Hill, R. A., 1991). Isoprenoids include hopane triterpenes, ubiquinones and menaquinones in bacteria, carotenoids, plastoquinones, mono-, sesqui-, di-, and tri-terpenes, and the prenyl side chains of chlorophylls in plants, and quinones, dolichols, steroids and retinoids in mammals (Edwards, P. A. & Ericcson, J. 1999).
Until recently it was generally assumed that IPP was derived solely from mevalonate synthesized from the condensation of three molecules of acetyl-CoA (McGarvey, D. J. & Croteau, R., 1995). However, recent independent studies demonstrated the existence of a novel, mevalonate-independent pathway for IPP synthesis known as the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DXP/MEP) pathway (Rohmer, M. et al., 1993; Rohmer, M., 1999; Schwender, J. et al., 1996; Eisenreich, W. et al., 1998). This latter mevalonate-independent pathway utilizes pyruvate and glyceraldehyde 3-phosphate as starting materials for production of IPP (Rohmer, M. et al., 1996) (FIG. 1).
Since vertebrates synthesize isoprenoid precursors using a mevalonate pathway, enzymes of the mevalonate-independent (DXP/MEP) pathway for isoprenoid production represent attractive targets for the structure-based design of selective pharmaceutical compounds. The DXP/MEP pathway occurs in a variety of eubacteria that includes several pathogenic species such as Mycobacterium tuberculosis, in algae (Rohmer, M., 1999), in the plastids of plant cells (Schwender, J. et al., 1999) and in the apicoplast of Plasmodium falciparum (the parasite that causes malaria) (Jomaa, H. et al., 1999; Vial, H. J., 2000). Given the essential nature of the DXP/MEP pathway in these organisms and the absence of this pathway in mammals, the enzymes comprising the DXP/MEP pathway represent unique targets for the generation of selective antibacterial (Rohmer, M., 1998; Kuzuyama, T. et al., 1998), antimalarial (Jomaa, H. et al., 1999; Vial, H. J., 2000; Ridley, R. G., 1999), and herbicidal (Lichtenthaler, H. K. et al., 2000) molecules.
For example, the YgbP protein of E. coli encodes the enzyme 4 diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthase (Rohdick, F. et al., 1999; Kuzuyama, T. et al., 2000). CDP-ME synthase belongs to the cytidyltransferase family of enzymes but utilizes a distinct architecture and a novel set of active site residues for CDP-ME formation. CDP-ME is a critical intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, in algae, in the plastids of plants, and in the malaria parasite, catalyzing the formation of CDP-ME from 2-C-methyl-D-erythritol-4-phosphate (Koppisch, A. T. et al., 2000) and cytidine triphosphate (CTP). Accordingly, there is a need in the art for the three dimensional protein structures of E. coli CDP-ME synthase and related proteins in order to reveal the stereochemical principles underlying substrate recognition and catalysis in CDP-ME synthase.